Exploring the predictive power of jejunal microbiome composition in clinical and subclinical necrotic enteritis caused by Clostridium perfringens: insights from a broiler chicken model.

BACKGROUND: Necrotic enteritis (NE) is a severe intestinal infection that affects both humans and poultry. It is caused by the bacterium Clostridium perfringens (CP), but the precise mechanisms underlying the disease pathogenesis remain elusive. This study aims to develop an NE broiler chicken model, explore the impact of the microbiome on NE pathogenesis, and study the virulence of CP isolates with different toxin gene combinations. METHODS: This study established an animal disease model for NE in broiler chickens. The methodology encompassed inducing abrupt protein changes and immunosuppression in the first experiment, and in the second, challenging chickens with CP isolates containing various toxin genes. NE was evaluated through gross and histopathological scoring of the jejunum. Subsequently, jejunal contents were collected from these birds for microbiome analysis via 16S rRNA amplicon sequencing, followed by sequence analysis to investigate microbial diversity and abundance, employing different bioinformatic approaches. RESULTS: Our findings reveal that CP infection, combined with an abrupt increase in dietary protein concentration and/or infection with the immunosuppressive variant infectious bursal disease virus (vIBDV), predisposed birds to NE development. We observed a significant decrease (p < 0.0001) in the abundance of Lactobacillus and Romboutsia genera in the jejunum, accompanied by a notable increase (p < 0.0001) in Clostridium and Escherichia. Jejunal microbial dysbiosis and severe NE lesions were particularly evident in birds infected with CP isolates containing cpa, netB, tpeL, and cpb2 toxin genes, compared to CP isolates with other toxin gene combinations. Notably, birds that did not develop clinical or subclinical NE following CP infection exhibited a significantly higher (p < 0.0001) level of Romboutsia. These findings shed light on the complex interplay between CP infection, the gut microbiome, and NE pathogenesis in broiler chickens. CONCLUSION: Our study establishes that dysbiosis within the jejunal microbiome serves as a reliable biomarker for detecting subclinical and clinical NE in broiler chicken models. Additionally, we identify the potential of the genera Romboutsia and Lactobacillus as promising candidates for probiotic development, offering effective alternatives to antibiotics in NE prevention and control.

Protein structural insights into a rare PCSK9 gain-of-function variant (R496W) causing familial hypercholesterolemia in a Saudi family: whole exome sequencing and computational analysis.

Familial hypercholesterolemia (FH) is a globally underdiagnosed genetic condition associated with premature cardiovascular death. The genetic etiology data on Arab FH patients is scarce. Therefore, this study aimed to identify the genetic basis of FH in a Saudi family using whole exome sequencing (WES) and multidimensional bioinformatic analysis. Our WES findings revealed a rare heterozygous gain-of-function variant (R496W) in the exon 9 of the PCSK9 gene as a causal factor for FH in this family. This variant was absent in healthy relatives of the proband and 200 healthy normolipidemic controls from Saudi Arabia. Furthermore, this variant has not been previously reported in various regional and global population genomic variant databases. Interestingly, this variant is classified as "likely pathogenic" (PP5) based on the variant interpretation guidelines of the American College of Medical Genetics (ACMG). Computational functional characterization suggested that this variant could destabilize the native PCSK9 protein and alter its secondary and tertiary structural features. In addition, this variant was predicted to negatively influence its ligand-binding ability with LDLR and Alirocumab antibody molecules. This rare PCSK9 (R496W) variant is likely to expand our understanding of the genetic basis of FH in Saudi Arabia. This study also provides computational structural insights into the genotype-protein phenotype relationship of PCSK9 pathogenic variants and contributes to the development of personalized medicine for FH patients in the future.

Potential Biomarkers for Parkinson Disease from Functional Enrichment and Bioinformatic Analysis of Global Gene Expression Patterns of Blood and Substantia Nigra Tissues.

The Parkinson disease (PD) is the second most common neurodegenerative disorder affecting the central nervous system and motor functions. The biological complexity of PD is yet to reveal potential targets for intervention or to slow the disease severity. Therefore, this study aimed to compare the fidelity of blood to substantia nigra (SN) tissue gene expression from PD patients to provide a systematic approach to predict role of the key genes of PD pathobiology. Differentially expressed genes (DEGs) from multiple microarray data sets of PD blood and SN tissue from GEO database are identified. Using the theoretical network approach and variety of bioinformatic tools, we prioritized the key genes from DEGs. A total of 540 and 1024 DEGs were identified in blood and SN tissue samples, respectively. Functional pathways closely related to PD such as ERK1 and ERK2 cascades, mitogen-activated protein kinase (MAPK) signaling, Wnt, nuclear factor-κB (NF-κB), and PI3K-Akt signaling were observed by enrichment analysis. Expression patterns of 13 DEGs were similar in both blood and SN tissues. Comprehensive network topological analysis and gene regulatory networks identified additional 10 DEGs functionally connected with molecular mechanisms of PD through the mammalian target of rapamycin (mTOR), autophagy, and AMP-activated protein kinase (AMPK) signaling pathways. Potential drug molecules were identified by chemical-protein network and drug prediction analysis. These potential candidates can be further validated in vitro/in vivo to be used as biomarkers and/or novel drug targets for the PD pathology and/or to arrest or delay the neurodegeneration over the years, respectively.

Anti-angiogenic effect of nano-formulated water soluble kaempferol and combretastatin in an in vivo chick chorioallantoic membrane model and HUVEC cells.

The present study evaluated the efficacy of nano-formulated water-soluble kaempferol and combretastatin alone and combined against the native kaempferol and combretastatin on angiogenesis. The solvent evaporation method was used to synthesize the nano-formulated water-soluble kaempferol and combretastatin and characterized using various analyses such as dynamic light scattering (DLS) and Fourier-transform infrared (FT-IR) spectroscopy.The anti-angiogenic activity of native, nano-formulated water-soluble kaempferol and combretastatin was investigated by cell viability on HUVEC and A498 cell lines, while chick chorioallantoic membrane (CAM) assay was utilized to assess morphometric and histopathological changes, and mRNA expressions of VEGF-A and FGF2 using qRT-PCR. MTT assay results revealed that the combination of nano-formulated water-soluble kaempferol and combretastatin significantly reduced the cell viability compared to control, individual treatments of native, nano-formulated water-soluble kaempferol, and combretastatin. Morphometric analysis of CAM showed that treatment with nano-formulated water-soluble kaempferol and combretastatin caused a substantial decrease in density, vessel network, branch points, and nets of CAM blood vessels. The histopathological results of CAM showed the irregular shape of blood vessels at the thin stratum of chronic endoderm, and blood capillaries were diminished compared to the control. In addition, the mRNA expression levels of VEGF-A and FGF2 were significantly decreased compared with native forms. Therefore, the findings of this study indicate that nano-formulated water-soluble combretastatin and kaempferol suppress angiogenesis by preventing the activation of endothelial cells and suppressing factors of angiogenesis. Moreover, a combination of nano-formulated water-soluble kaempferol and combretastatin worked much better than individual treatments.

Paget's disease: a review of the epidemiology, etiology, genetics, and treatment.

Paget's disease of bone (PDB) is the second most prevalent metabolic bone disorder worldwide, with a prevalence rate of 1.5%-8.3%. It is characterized by localized areas of accelerated, disorganized, and excessive bone production and turnover. Typically, PDB develops in the later stages of life, particularly in the late 50s, and affects men more frequently than women. PDB is a complex disease influenced by both genetic and environmental factors. PDB has a complex genetic basis involving multiple genes, with SQSTM1 being the gene most frequently associated with its development. Mutations affecting the UBA domain of SQSTM1 have been detected in both familial and sporadic PDB cases, and these mutations are often associated with severe clinical expression. Germline mutations in other genes such as TNFRSF11A, ZNF687 and PFN1, have also been associated with the development of the disease. Genetic association studies have also uncovered several PDB predisposing risk genes contributing to the disease pathology and severity. Epigenetic modifications of genes involved in bone remodelling and regulation, including RANKL, OPG, HDAC2, DNMT1, and SQSTM1, have been implicated in the development and progression of Paget's disease of bone, providing insight into the molecular basis of the disease and potential targets for therapeutic intervention. Although PDB has a tendency to cluster within families, the variable severity of the disease across family members, coupled with decreasing incidence rates, indicates that environmental factors may also play a role in the pathophysiology of PDB. The precise nature of these environmental triggers and how they interact with genetic determinants remain poorly understood. Fortunately, majority of PDB patients can achieve long-term remission with an intravenous infusion of aminobisphosphonates, such as zoledronic acid. In this review, we discuss aspects like clinical characteristics, genetic foundation, and latest updates in PDB research.

Rare variant burden analysis from exomes of three consanguineous families reveals LILRB1 and PRSS3 as potential key proteins in inflammatory bowel disease pathogenesis.

Background: Inflammatory bowel disease (IBD) is a chronic autoimmune disorder characterized by severe inflammation and mucosal destruction of the intestine. The specific, complex molecular processes underlying IBD pathogenesis are not well understood. Therefore, this study is aimed at identifying and uncovering the role of key genetic factors in IBD. Method: The whole exome sequences (WESs) of three consanguineous Saudi families having many siblings with IBD were analyzed to discover the causal genetic defect. Then, we used a combination of artificial intelligence approaches, such as functional enrichment analysis using immune pathways and a set of computational functional validation tools for gene expression, immune cell expression analyses, phenotype aggregation, and the system biology of innate immunity, to highlight potential IBD genes that play an important role in its pathobiology. Results: Our findings have shown a causal group of extremely rare variants in the LILRB1 (Q53L, Y99N, W351G, D365A, and Q376H) and PRSS3 (F4L and V25I) genes in IBD-affected siblings. Findings from amino acids in conserved domains, tertiary-level structural deviations, and stability analysis have confirmed that these variants have a negative impact on structural features in the corresponding proteins. Intensive computational structural analysis shows that both genes have very high expression in the gastrointestinal tract and immune organs and are involved in a variety of innate immune system pathways. Since the innate immune system detects microbial infections, any defect in this system could lead to immune functional impairment contributing to IBD. Conclusion: The present study proposes a novel strategy for unraveling the complex genetic architecture of IBD by integrating WES data of familial cases, with computational analysis.

Combining machine learning and structure-based approaches to develop oncogene PIM kinase inhibitors.

Introduction: PIM kinases are targets for therapeutic intervention since they are associated with a number of malignancies by boosting cell survival and proliferation. Over the past years, the rate of new PIM inhibitors discovery has increased significantly, however, new generation of potent molecules with the right pharmacologic profiles were in demand that can probably lead to the development of Pim kinase inhibitors that are effective against human cancer. Method: In the current study, a machine learning and structure based approaches were used to generate novel and effective chemical therapeutics for PIM-1 kinase. Four different machine learning methods, namely, support vector machine, random forest, k-nearest neighbour and XGBoost have been used for the development of models. Total, 54 Descriptors have been selected using the Boruta method. Results: SVM, Random Forest and XGBoost shows better performance as compared to k-NN. An ensemble approach was implemented and, finally, four potential molecules (CHEMBL303779, CHEMBL690270, MHC07198, and CHEMBL748285) were found to be effective for the modulation of PIM-1 activity. Molecular docking and molecular dynamic simulation corroborated the potentiality of the selected molecules. The molecular dynamics (MD) simulation study indicated the stability between protein and ligands. Discussion: Our findings suggest that the selected models are robust and can be potentially useful for facilitating the discovery against PIM kinase.

A comparative mRNA- and miRNA transcriptomics reveals novel molecular signatures associated with metastatic prostate cancers.

Background: Prostate cancer (PC) is a fatally aggressive urogenital cancer killing millions of men, globally. Thus, this study aims to identify key miRNAs, target genes, and drug targets associated with prostate cancer metastasis. Methods: The miRNA and mRNA expression datasets of 148 prostate tissue biopsies (39 tumours and 109 normal tissues), were analysed by differential gene expression analysis, protein interactome mapping, biological pathway analysis, miRNA-mRNA networking, drug target analysis, and survival curve analysis. Results: The dysregulated expression of 53 miRNAs and their 250 target genes involved in Hedgehog, ErbB, and cAMP signalling pathways connected to cell growth, migration, and proliferation of prostate cancer cells was detected. The subsequent miRNA-mRNA network and expression status analysis have helped us in narrowing down their number to 3 hub miRNAs (hsa-miR-455-3p, hsa-miR-548c-3p, and hsa-miR-582-5p) and 9 hub genes (NFIB, DICER1, GSK3B, DCAF7, FGFR1OP, ABHD2, NACC2, NR3C1, and FGF2). Further investigations with different systems biology methods have prioritized NR3C1, ABHD2, and GSK3B as potential genes involved in prostate cancer metastasis owing to their high mutation load and expression status. Interestingly, down regulation of NR3C1 seems to improve the prostate cancer patient survival rate beyond 150 months. The NR3C1, ABHD2, and GSK3B genes are predicted to be targeted by hsa-miR-582-5p, besides some antibodies, PROTACs and inhibitory molecules. Conclusion: This study identified key miRNAs (miR-548c-3p and miR-582-5p) and target genes (NR3C1, ABHD2, and GSK3B) as potential biomarkers for metastatic prostate cancers from large-scale gene expression data using systems biology approaches.

Structural characterization and conformational dynamics of alpha-1 antitrypsin pathogenic variants causing alpha-1-antitrypsin deficiency.

Background: Alpha-1 antitrypsin deficiency (A1ATD) is a progressive lung disease caused by inherited pathogenic variants in the SERPINA1 gene. However, their actual role in maintenance of structural and functional characteristics of the corresponding α-1 anti-trypsin (A1AT) protein is not well characterized. Methods: The A1ATD causative SERPINA1 missense variants were initially collected from variant databases, and they were filtered based on their pathogenicity potential. Then, the tertiary protein models were constructed and the impact of individual variants on secondary structure, stability, protein-protein interactions, and molecular dynamic (MD) features of the A1AT protein was studied using diverse computational methods. Results: We identified that A1ATD linked SERPINA1 missense variants like F76S, S77F, L278P, E288V, G216C, and H358R are highly deleterious as per the consensual prediction scores of SIFT, PolyPhen, FATHMM, M-CAP and REVEL computational methods. All these variants were predicted to alter free energy dynamics and destabilize the A1AT protein. These variants were seen to cause minor structural drifts at residue level (RMSD = <2Å) of the protein. Interestingly, S77F and L278P variants subtly alter the size of secondary structural elements like beta pleated sheets and loops. The residue level fluctuations at 100 ns simulation confirm the highly damaging structural consequences of all the six missense variants on the conformation dynamics of the A1AT protein. Moreover, these variants were also predicted to cause functional deformities by negatively impacting the binding energy of A1AT protein with NE ligand molecule. Conclusion: This study adds a new computational biology dimension to interpret the genotype-protein phenotype relationship between SERPINA1 pathogenic variants with its structural plasticity and functional behavior with NE ligand molecule contributing to the Alpha-1-antitrypsin deficiency. Our results support that A1ATD complications correlates with the conformational flexibility and its propensity of A1AT protein polymerization when misfolded.

Identification of miRNA-mRNA-TFs regulatory network and crucial pathways involved in asthma through advanced systems biology approaches.

Asthma is a life-threatening and chronic inflammatory lung disease that is posing a true global health challenge. The genetic basis of the disease is fairly well examined. However, the molecular crosstalk between microRNAs (miRNAs), target genes, and transcription factors (TFs) networks and their contribution to disease pathogenesis and progression is not well explored. Therefore, this study was aimed at dissecting the molecular network between mRNAs, miRNAs, and TFs using robust computational biology approaches. The transcriptomic data of bronchial epithelial cells of severe asthma patients and healthy controls was studied by different systems biology approaches like differentially expressed gene detection, functional enrichment, miRNA-target gene pairing, and mRNA-miRNA-TF molecular networking. We detected the differential expression of 1703 (673 up-and 1030 down-regulated) genes and 71 (41 up-and 30 down-regulated) miRNAs in the bronchial epithelial cells of asthma patients. The DEGs were found to be enriched in key pathways like IL-17 signaling (KEGG: 04657), Th1 and Th2 cell differentiation (KEGG: 04658), and the Th17 cell differentiation (KEGG: 04659) (p-values = 0.001). The results from miRNAs-target gene pairs-transcription factors (TFs) have detected the key roles of 3 miRs (miR-181a-2-3p; miR-203a-3p; miR-335-5p), 6 TFs (TFAM, FOXO1, GFI1, IRF2, SOX9, and HLF) and 32 miRNA target genes in eliciting autoimmune reactions in bronchial epithelial cells of the respiratory tract. Through systemic implementation of comprehensive system biology tools, this study has identified key miRNAs, TFs, and miRNA target gene pairs as potential tissue-based asthma biomarkers.

Granulomatous hepatitis in a Saudi child with IL2RA defect: a case report and literature review.

Interleukin-2 receptor alpha (IL2RA) defect (OMIM- # 606367) is an immune disease where affected patients are vulnerable to developing recurrent microbial infections in addition to lymphadenopathy and dermatological manifestations. This condition is known to be caused by pathogenic variants in the IL2RA gene, which are inherited in an autosomal recessive fashion. In this case report, we present a patient with IL2RA defect from Saudi Arabia who presented with chronic diarrhea, poor weight gain, mild villous atrophy, malnutrition, hepatomegaly, nonspecific inflammation, and an eczematous skin rash. His genetic analysis revealed a novel, homozygous, and likely pathogenic variant, that is, c.504 C>A (Cys168Ter), located in the exon 4of the IL2RA gene, which was inherited from his parents in an autosomal recessive mode of inheritance. This variant produces a 272-amino-acid shorter IL2RA protein chain, which most likely becomes degraded in the cytosol. Thus, we assume that the c.504 C>A is a null allele that abolishes the synthesis of IL2RA, malforms the IL-2 receptor complex, and eventually causes immunodeficiency manifestations. To our knowledge, this is the first time a person with IL2RA defect has shown signs of granulomatous hepatitis on a liver biopsy.

Complex Inheritance of Rare Missense Variants in PAK2, TAP2, and PLCL1 Genes in a Consanguineous Arab Family With Multiple Autoimmune Diseases Including Celiac Disease.

Background: Autoimmune diseases (AIDs) share a common molecular etiology and often present overlapping clinical presentations. Thus, this study aims to explore the complex molecular basis of AID by whole exome sequencing and computational biology analysis. Methods: Molecular screening of the consanguineous AID family and the computational biology characterization of the potential variants were performed. The potential variants were searched against the exome data of 100 healthy individuals and 30 celiac disease patients. Result: A complex inheritance pattern of PAK2 (V43A), TAP2 (F468Y), and PLCL1 (V473I) genetic variants was observed in the three probands of the AID family. The PAK2 variant (V43A) is a novel one, but TAP2 (F468Y) and PLCL1 (V473I) variants are extremely rare in local Arab (SGHP and GME) and global (gnomAD) databases. All these variants were localized in functional domains, except for the PAK2 variant (V43A) and were predicted to alter the structural (secondary structure elements, folding, active site confirmation, stability, and solvent accessibility) and functional (gene expression) features. Therefore, it is reasonable to postulate that the dysregulation of PAK2, TAP2, and PLCL1 genes is likely to elicit autoimmune reactions by altering antigen processing and presentation, T cell receptor signaling, and immunodeficiency pathways. Conclusion: Our findings highlight the importance of exploring the alternate inheritance patterns in families presenting complex autoimmune diseases, where classical genetic models often fail to explain their molecular basis. These findings may have potential implications for developing personalized therapies for complex disease patients.

Exome Sequencing Identifies the Extremely Rare ITGAV and FN1 Variants in Early Onset Inflammatory Bowel Disease Patients.

Background: Molecular diagnosis of early onset inflammatory bowel disease (IBD) is very important for adopting suitable treatment strategies. Owing to the sparse data available, this study aims to identify the molecular basis of early onset IBD in Arab patients. Methods: A consanguineous Arab family with monozygotic twins presenting early onset IBD was screened by whole exome sequencing (WES). The variants functional characterization was performed by a series of computational biology methods. The IBD variants were further screened in in-house whole exome data of 100 Saudi cohorts ensure their rare prevalence in the population. Results: Genetic screening has identified the digenic autosomal recessive mode of inheritance of ITGAV (G58V) and FN1 (G313V) variants in IBD twins with early onset IBD. Findings from pathogenicity predictions, stability and molecular dynamics have confirmed the deleterious nature of both variants on structural features of the corresponding proteins. Functional biology data suggested that both genes show abundant expression in gastrointestinal tract and immune organs, involved in immune cell restriction, regulation of different immune related pathways. Data from knockout mouse models for ITGAV gene has revealed that the dysregulated expression of this gene impacts intestinal immune homeostasis. The defective ITGAV and FN1 involved in integrin pathway, are likely to induce intestinal inflammation by disturbing immune homeostasis. Conclusions: Our findings provide novel insights into the molecular etiology of pediatric onset IBD and may likely pave way in developing genomic medicine.

Genome-Wide Association Study-Guided Exome Rare Variant Burden Analysis Identifies IL1R1 and CD3E as Potential Autoimmunity Risk Genes for Celiac Disease.

Celiac disease (CeD) is a multifactorial autoimmune enteropathy characterized by the overactivation of the immune system in response to dietary gluten. The molecular etiology of CeD is still not well-understood. Therefore, this study aims to identify potential candidate genes involved in CeD pathogenesis by applying multilayered system biology approaches. Initially, we identified rare coding variants shared between the affected siblings in two rare Arab CeD families by whole-exome sequencing (WES). Then we used the STRING database to construct a protein network of rare variants and genome-wide association study (GWAS) loci to explore their molecular interactions in CeD. Furthermore, the hub genes identified based on network topology parameters were subjected to a series of computational validation analyses like pathway enrichment, gene expression, knockout mouse model, and variant pathogenicity predictions. Our findings have shown the absence of rare variants showing classical Mendelian inheritance in both families. However, interactome analysis of rare WES variants and GWAS loci has identified a total of 11 hub genes. The multidimensional computational analysis of hub genes has prioritized IL1R1 for family A and CD3E for family B as potential genes. These genes were connected to CeD pathogenesis pathways of T-cell selection, cytokine signaling, and adaptive immune response. Future multi-omics studies may uncover the roles of IL1R1 and CD3E in gluten sensitivity. The present investigation lays forth a novel approach integrating next-generation sequencing (NGS) of familial cases, GWAS, and computational analysis for solving the complex genetic architecture of CeD.

Bioinformatics insights into the genes and pathways on severe COVID-19 pathology in patients with comorbidities.

Background: Coronavirus disease (COVID-19) infection is known for its severe clinical pathogenesis among individuals with pre-existing comorbidities. However, the molecular basis of this observation remains elusive. Thus, this study aimed to map key genes and pathway alterations in patients with COVID-19 and comorbidities using robust systems biology approaches. Methods: The publicly available genome-wide transcriptomic datasets from 120 COVID-19 patients, 281 patients suffering from different comorbidities (like cardiovascular diseases, atherosclerosis, diabetes, and obesity), and 252 patients with different infectious diseases of the lung (respiratory syncytial virus, influenza, and MERS) were studied using a range of systems biology approaches like differential gene expression, gene ontology (GO), pathway enrichment, functional similarity, mouse phenotypic analysis and drug target identification. Results: By cross-mapping the differentially expressed genes (DEGs) across different datasets, we mapped 274 shared genes to severe symptoms of COVID-19 patients or with comorbidities alone. GO terms and functional pathway analysis highlighted genes in dysregulated pathways of immune response, interleukin signaling, FCGR activation, regulation of cytokines, chemokines secretion, and leukocyte migration. Using network topology parameters, phenotype associations, and functional similarity analysis with ACE2 and TMPRSS2-two key receptors for this virus-we identified 17 genes with high connectivity (CXCL10, IDO1, LEPR, MME, PTAFR, PTGS2, MAOB, PDE4B, PLA2G2A, COL5A1, ICAM1, SERPINE1, ABCB1, IL1R1, ITGAL, NCAM1 and PRKD1) potentially contributing to the clinical severity of COVID-19 infection in patients with comorbidities. These genes are predicted to be tractable and/or with many existing approved inhibitors, modulators, and enzymes as drugs. Conclusion: By systemic implementation of computational methods, this study identified potential candidate genes and pathways likely to confer disease severity in COVID-19 patients with pre-existing comorbidities. Our findings pave the way to develop targeted repurposed therapies in COVID-19 patients.

Computational approaches for discovering significant microRNAs, microRNA-mRNA regulatory pathways, and therapeutic protein targets in endometrial cancer.

Endometrial cancer (EC) is a urogenital cancer affecting millions of post-menopausal women, globally. This study aims to identify key miRNAs, target genes, and drug targets associated with EC metastasis. The global miRNA and mRNA expression datasets of endometrial tissue biopsies (24 tumors +3 healthy tissues for mRNA and 18 tumor +4 healthy tissues for miRNAs), were extensively analyzed by mapping of DEGs, DEMi, biological pathway enrichment, miRNA-mRNA networking, drug target identification, and survival curve output for differentially expressed genes. Our results reveal the dysregulated expression of 26 miRNAs and their 66 target genes involved in focal adhesions, p53 signaling pathway, ECM-receptor interaction, Hedgehog signaling pathway, fat digestion and absorption, glioma as well as retinol metabolism involved in cell growth, migration, and proliferation of endometrial cancer cells. The subsequent miRNA-mRNA network and expression status analysis have narrowed down to 2 hub miRNAs (hsa-mir-200a, hsa-mir-429) and 6 hub genes (PTCH1, FOSB, PDGFRA, CCND2, ABL1, ALDH1A1). Further investigations with different systems biology methods have prioritized ALDH1A1, ABL1 and CCND2 as potential genes involved in endometrial cancer metastasis owing to their high mutation load and expression status. Interestingly, overexpression of PTCH1, ABL1 and FOSB genes are reported to be associated with a low survival rate among cancer patients. The upregulated hsa-mir-200a-b is associated with the decreased expression of the PTCH1, CCND2, PDGFRA, FOSB and ABL1 genes in endometrial cancer tissue while hsa-mir-429 is correlated with the decreased expression of the ALDH1A1 gene, besides some antibodies, PROTACs and inhibitory molecules. In conclusion, this study identified key miRNAs (hsa-mir-200a, hsa-mir-429) and target genes ALDH1A1, ABL1 and CCND2 as potential biomarkers for metastatic endometrial cancers from large-scale gene expression data using systems biology approaches.

Novel MYO1D Missense Variant Identified Through Whole Exome Sequencing and Computational Biology Analysis Expands the Spectrum of Causal Genes of Laterality Defects.

Laterality defects (LDs) or asymmetrically positioned organs are a group of rare developmental disorders caused by environmental and/or genetic factors. However, the exact molecular pathophysiology of LD is not yet fully characterised. In this context, studying Arab population presents an ideal opportunity to discover the novel molecular basis of diseases owing to the high rate of consanguinity and genetic disorders. Therefore, in the present study, we studied the molecular basis of LD in Arab patients, using next-generation sequencing method. We discovered an extremely rare novel missense variant in MYO1D gene (Pro765Ser) presenting with visceral heterotaxy and left isomerism with polysplenia syndrome. The proband in this index family has inherited this homozygous variant from her heterozygous parents following the autosomal recessive pattern. This is the first report to show MYO1D genetic variant causing left-right axis defects in humans, besides previous known evidence from zebrafish, frog and Drosophila models. Moreover, our multilevel bioinformatics-based structural (protein variant structural modelling, divergence, and stability) analysis has suggested that Ser765 causes minor structural drifts and stability changes, potentially affecting the biophysical and functional properties of MYO1D protein like calmodulin binding and microfilament motor activities. Functional bioinformatics analysis has shown that MYO1D is ubiquitously expressed across several human tissues and is reported to induce severe phenotypes in knockout mouse models. In conclusion, our findings show the expanded genetic spectrum of LD, which could potentially pave way for the novel drug target identification and development of personalised medicine for high-risk families.

TagSNP approach for HLA risk allele genotyping of Saudi celiac disease patients: effectiveness and pitfalls.

BACKGROUND: Celiac disease (CD) is a genetically complex autoimmune disease which is triggered by dietary gluten. Human leukocyte antigen (HLA) class II genes are known to act as high-risk markers for CD, where >95% of CD patients carry (HLA), DQ2 and/or DQ8 alleles. Therefore, the present study was conducted to investigate the distribution of HLA haplotypes among Saudi CD patients and healthy controls by using the tag single nucleotide polymorphisms (SNP). METHODS: HLA-tag SNPs showing strong linkage value (r2>0.99) were used to predict the HLA DQ2 and DQ8 genotypes in 101 Saudi CD patients and in 103 healthy controls by using real-time polymerase chain reaction technique. Genotype calls were further validated by Sanger sequencing method. RESULTS: A total of 63.7% of CD cases and of 60.2% of controls were predicted to carry HLA-DQ2 and DQ8 heterodimers, either in the homozygous or heterozygous states. The prevalence of DQ8 in our CD patients was predicted to be higher than the patients from other ethnic populations (35.6%). More than 32% of the CD patients were found to be non-carriers of HLA risk haplotypes as predicted by the tag SNPs. CONCLUSION: The present study highlights that the Caucasian specific HLA-tag SNPs would be of limited value to accurately predict CD specific HLA haplotypes in Saudi population, when compared with the Caucasian groups. Prediction of risk haplotypes by tag SNPs in ethnic groups is a good alternate approach as long as the tag SNPs were identified from the local population genetic variant databases.

Identification of a Rare Exon 19 Skipping Mutation in ALMS1 Gene in Alström Syndrome Patients From Two Unrelated Saudi Families.

Background: Alström syndrome (AS) is a very rare childhood disorder characterized by cardiomyopathy, progressive hearing loss and blindness. Inherited genetic variants of ALMS1 gene are the known molecular cause of this disease. The objective of this study was to characterize the genetic basis and understand the genotype-phenotype relationship in Saudi AS patients. Methods: Clinical phenotyping and whole-exome sequencing (WES) analysis were performed on six AS patients belonging to two unrelated consanguineous Saudi families. Sanger sequencing was performed to determine the mode of inheritance of ALMS1 variant in first-degree family relatives and also to ensure its rare prevalence in 100 healthy population controls. Results: We identified that Alström patients from both the families were sharing a very rare ALMS1, 3'-splice site acceptor (c.11873-2 A>T) variant, which skips entire exon-19 and shortens the protein by 80 amino acids. This disease variant was inherited by AS patients in autosomal recessive mode and is not yet reported in any population-specific genetic databases. AS patients carrying this mutation showed heterogeneity in clinical presentations. Computational analysis of the mutant centroid structure of ALMS1 mRNA revealed that exon-19 skipping enlarges the hairpin loop and decreases the free energy, eventually affecting its folding pattern, stability, and function. Hence, we propose c.11873-2A as an AS causative potential founder mutation in Saudi Arabia because it is found in two families lacking a common lineage. Conclusions: We conclude that WES analysis potentially helps in clinical phenotyping, early diagnosis, and better clinical management of Alström patients showing variable clinical expressivity.

Molecular differential analysis of uterine leiomyomas and leiomyosarcomas through weighted gene network and pathway tracing approaches.

Uterine smooth muscular neoplastic growths like benign leiomyomas (UL) and metastatic leiomyosarcomas (ULMS) share similar clinical symptoms, radiological and histological appearances making their clinical distinction a difficult task. Therefore, the objective of this study is to identify key genes and pathways involved in transformation of UL to ULMS through molecular differential analysis. Global gene expression profiles of 25 ULMS, 25 UL, and 29 myometrium (Myo) tissues generated on Affymetrix U133A 2.0 human genome microarrays were analyzed by deploying robust statistical, molecular interaction network, and pathway enrichment methods. The comparison of expression signals across Myo vs UL, Myo vs ULMS, and UL vs ULMS groups identified 249, 1037, and 716 significantly expressed genes, respectively (p ≤ 0.05). The analysis of 249 DEGs from Myo vs UL confirms multistage dysregulation of various key pathways in extracellular matrix, collagen, cell contact inhibition, and cytokine receptors transform normal myometrial cells to benign leiomyomas (p value ≤ 0.01). The 716 DEGs between UL vs ULMS were found to affect cell cycle, cell division related Rho GTPases and PI3K signaling pathways triggering uncontrolled growth and metastasis of tumor cells (p value ≤ 0.01). Integration of gene networking data, with additional parameters like estimation of mutation burden of tumors and cancer driver gene identification, has led to the finding of 4 hubs (JUN, VCAN, TOP2A, and COL1A1) and 8 bottleneck genes (PIK3R1, MYH11, KDR, ESR1, WT1, CCND1, EZH2, and CDKN2A), which showed a clear distinction in their distribution pattern among leiomyomas and leiomyosarcomas. This study provides vital clues for molecular distinction of UL and ULMS which could further assist in identification of specific diagnostic markers and therapeutic targets.Abbreviations UL: Uterine Leiomyomas; ULMS: Uterine Leiomyosarcoma; Myo: Myometrium; DEGs: Differential Expressed Genes; RMA: Robust Multiarray Average; DC: Degree of Centrality; BC: Betweenness of Centrality; CGC: Cancer Gene Census; FDR: False Discovery Rate; TCGA: Cancer Genome Atlas; BP: Biological Process; CC: Cellular Components; MF: Molecular Function; PPI: Protein-Protein Interaction.